MUSCARINIC SIGNLING: REGULATION OF VENTRICULAR FUNCTION

描述（由申请人提供）：心脏的机械性能由其固有的收缩特性决定，并受到自主神经系统的平衡调节。虽然α-肾上腺素能刺激增加泵功能，但胆碱能刺激通过降低先前由cAMP升高的L-型Ca 2+通道（ICa-L）的电导来降低泵功能（“增强的拮抗作用”）。两种不同的G蛋白，Go和Gi-2，被认为是这种效应绝对需要的，但它们的确切作用和相互作用以及所涉及的信号传导机制仍然不清楚。我们有初步的数据表明，Go蛋白也可能通过改变兴奋收缩（E-C）偶联和肌丝对Ca 2+的反应来调节Ca 2+循环和力的产生。本研究的总体目标是定义将Go和/或Gi 2蛋白激活与心室Ca 2+通量和收缩功能的毒蕈碱调节联系起来的信号传导机制，并测试选择性干扰该途径可用于增强体内收缩功能的假设。具体目标是：（1）为了验证激活的Gao的表达通过增加心肌细胞内Ca 2+浓度来调节心肌细胞内Ca 2+循环和细胞缩短的假设，（而不是降低）E-C偶联增益和肌丝对Ca 2+的反应性，与其对ICa-L的钝化作用一致，（2）测试电池是否-渗透性肽输入可适于将显性负性抑制肽引入成年心室心肌细胞，作为替代和可能的改进，腺病毒基因转移，（3）检验Go和Gi 2蛋白协同作为M2受体对ICa-L、Ca 2+循环和收缩性作用的介导物，以及G α和G β γ均有助于这种作用的假设，（4）检验在心室心肌中干扰Go和/或Gi 2介导的信号转导可用于增强心室收缩功能的假设。毒蕈碱“增强的拮抗作用”在正常和病理生理条件下都具有生理重要性。更好地了解毒蕈碱受体介导的β-肾上腺素能刺激和收缩功能钝化的反对所涉及的过程可能形成的基础上，治疗干预措施，旨在长期自主输入心脏通过一个迄今未充分探索的机制。

DESCRIPTION (provided by applicant): The mechanical performance of the heart is determined by its intrinsic contractile properties and is subject to counterbalancing regulation by the autonomic nervous system. While a-adrenergic stimulation increases pump function, cholinergic stimulation decreases it by reducing the conductance of the L-type Ca2+ channel (ICa-L) previously elevated by cAMP ("accentuated antagonism"). Two different G proteins, Go and Gi-2, have been implicated to be absolutely required for this effect, but their exact role and interplay as well as the signaling mechanisms involved are still poorly defined. We have preliminary data suggesting that Go protein may also regulate Ca2+ cycling and force generation by altering excitation contraction (E-C) coupling and the responsiveness of the myofilaments to Ca2+. The overall goal of this investigation is to define the signaling mechanisms that link Go and/or Gi2 protein activation to the muscarinic regulation of ventricular Ca2+ fluxes and contractile function and to test the hypothesis that selective interference with this pathway can be utilized to enhance contractile function in vivo. The Specific Aims are: (1) to test the hypothesis that expression of activated Gao regulates Ca2+ cycling and cell shortening in the ventricular myocardium by increasing (rather than decreasing) E-C coupling gain and myofilament responsiveness to Ca2+ in concert to its blunting effect on ICa-L, (2) to test whether cell-permeable peptide import can be adapted for introduction of dominant negative inhibitory peptides into adult ventricular cardiocytes as an alternative and possible improvement to adenoviral gene transfer, (3) to test the hypotheses that Go and Gi2 proteins act in concert as mediators of M2-receptor effects on ICa-L, Ca2+ cycling and contractility and that both Galpha and Gbetagamma contribute to this effect (4) to test the hypothesis that interference with Go and/or Gi-mediated signal transduction in the ventricular myocardium can be utilized to enhance ventricular contractile function. Muscarinic "accentuated antagonism" is of physiological importance both under normal and pathophysiological conditions. A better understanding of the processes involved in muscarinic receptor-mediated opposition of beta-adrenergic stimulation and blunting of contractile function may form the basis for therapeutic interventions aimed at the long-term autonomic input to the heart through a so far underexplored mechanism.